Project Summary MHC class I-restricted antigen processing is essential for making CD8+ T cell responses. It must function correctly for effective immune recognition of pathogens, and aberrations can lead to autoimmunity. The overall aim of this proposal is to understand the quality control processes regulating MHC class I- restricted antigen processing of conventional antigens, translated in the cytosol, and of exogenous antigens internalized by cross-presenting cells. The latter process is particularly poorly characterized, yet it is essential for priming CD8+ T cell responses. We wish to understand how these two different processing mechanisms drive the assembly of essentially the same pool of MHC-I peptide complexes. A critical quality control process in the endoplasmic reticulum (ER) that remains ill understood is the action of the enzyme UDP-glucose glycoprotein transferase (UGT1), which produces the correct monoglucosylated glycan structure that allows MHC-I molecules to maintain an interaction with the Peptide Loading Complex (PLC) via the lectin chaperone calreticulin, facilitating tapasin-mediated peptide exchange. This promotes the expression by the cell of complexes of MHC-I with peptides of high affinity. We will test the hypothesis that structural flexibility in the MHC-I peptide binding domain serves as a recognition signal for UGT1. A second component, which is found in both the ER and the endocytic pathway, is the tapasin homologue TAPBPR. In vitro, TAPBPR can induce peptide exchange by MHC-I molecules outside of the PLC, and it has also been shown to interact with UGT1. A second major goal is to determine whether TAPBPR mediates peptide exchange by MHC class I molecules in intact cells, whether this can occur both in the ER and the phagosomes of cross-presenting cells, and whether in either of these intracellular compartments the simultaneous interaction of TAPBPR with UGT1 focuses the enzymatic activity of UGT1 onto MHC-I molecules associated with low affinity peptides, facilitating their exchange for optimal peptides. The final goal is to determine how cross-presented intact antigens encounter proteasomes, which are essential for both conventional MHC-I antigen processing and cross- presentation. We have evidence that this interaction occurs proximal to, and probably within, phagosomes of cross-presenting cells. The mechanisms that regulate this interaction at both the cell biological and biochemical level will be investigated.